Effect of Ph on Antibody Adsorption onto Gold Nanoparticles for Use in Biosensors
Gold nanoparticles (AuNPs) functionalized with antibodies have the potential to improve biosensing technology due to the unique optical properties of AuNPs and the specificity of antibody-antigen interactions. Critical to the development and optimization of these AuNP-enabled sensing technologies is the immobilization of the antibody onto the AuNP. Ideally, antibody-AuNPs conjugates must be stable in high salt environments, and the antibody-AuNP interaction must be sufficiently strong that when exposed to biological samples, the immobilized antibody will not be replaced by the abundant matrix proteins. In this research, direct adsorption of anti-HRP antibody to AuNPs was investigated as a function of pH. Nanoparticle tracking analysis (NTA) allows our group to investigate antibody adsorption on AuNP by measuring the increase in hydrodynamic diameter (Dh) of the AuNPs at different anti-HRP concentrations. Antibody adsorption data gathered from the NTA was modeled by the Hill-modified Langmuir adsorption isotherm to determine the maximum increase in Dh at each pH. Initial experiments suggest a monolayer of antibody is formed at saturation at each pH; however, the Dh increased by 13.4 nm at pH 7.5 and the Dh increased by 8.0 nm at pH 8.5. This data may suggest that pH affects antibody orientation. Our group developed an enzyme-mediated assay to quantify the amount of antibody that is available for antigen binding to gain further insight into antibody orientation. At pH 7.50, 45% of the immobilized antibodies were active and at pH 8.5 only 14% of the immobilized antibodies were active. Based on the Dh and activity values, this data suggests that at pH 8.5 the anti-HRP is lying flat on the AuNPs and at pH 7.5 the tail portion (Fc domain) of the anti-HRP is binding to the AuNPs, allowing the antigen to bind to the anti-HRP binding site (Fab domain). Ultimately, these studies aimed to elucidate the effect of pH on antibody adsorption onto AuNP to maximize antigen-binding activity of antibody-AuNP conjugates and enhance the performance of biosensing technologies.
Ruiz, Guadalupe, "Effect of Ph on Antibody Adsorption onto Gold Nanoparticles for Use in Biosensors" (2018). University Research Symposium. 131.